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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2004 as doi:10.1096/fj.03-1161fje. |
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,1
* Department of Anatomy I, Asahikawa Medical College, Asahikawa, Hokkaido, Japan;
Department of Pathology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Higashinari, Osaka, Japan;
Department of Anatomy and Neuroscience, Osaka University Medical School, Yamada Oka, Japan;
|| Division of Nephrology, Department of Medicine, Osaka Rosai Hospital, Sakai, Japan;
Department of Neuroscience, Kanazawa University Medical School, Kanazawa City, Ishikawa, Japan; and
¶ Dean’s Office, Medical College of Georgia, Augusta, Georgia, USA
2Correspondence: Department of Anatomy I, Asahikawa Medical College, Midorigaoka-Higashi 2-1-1-1, Asahikawa, 078-8510, Hokkaido, Japan. E-mail: ybando{at}asahikawa-med.ac.jp
SPECIFIC AIMS
Renal cell injury caused by ischemia/reperfusion (I/R) is often accompanied by acute failure of renal function, which is clinically of importance due to high mortality. 150 kDa oxygen-regulated protein (ORP150) is an inducible endoplasmic reticulum (ER) chaperone with cytoprotective properties in settings of cell stress, such as ischemia/reperfusion (I/R). Based upon the cytoprotective properties of 150 kDa oxygen regulated protein (ORP150) in ischemic condition, we have examined the role of ORP150 in renal ischemia/reperfusion (I/R).
PRINCIPAL FINDINGS
1. ORP150 is expressed in renal epithelial cells in both human and rat kidney after I/R
In acute tubular necrosis accompanying cardiogenic shock, ORP150 was detected mainly in parts of renal tubules in the cortex, and, more frequently, in the medulla. The same pattern of ORP150 expression was found in a case of osmotic nephrosis due to treatment of brain edema.
To further analyze expression of ORP150, rats were subjected to renal I/R by unilateral occlusion of the renal artery. Northern blot showed a marked increase in ORP150 transcripts after I/R on the ipsilateral side, peaking 8–12 h after reperfusion (Fig. 1
A). ORP150 transcripts were also induced on the contralateral side, though to a lesser extent (Fig. 1B
). In situ hybridization of normal kidney revealed a diffuse distribution of ORP150 transcripts in the medulla (Fig. 1C
, 1G
). ORP150 transcripts were strongly induced 12 h after I/R in the outer medulla, the area between cortex and medulla (Fig. 1D
, 1E
, 1H
, 1I
). Distribution of ORP150 transcripts overlapped, at least partially, with that observed for Tamm-Horsefall protein (THP) mRNA (Fig. 1F
), a marker of the thick ascending loop (TAL). Immunohistochemical analysis of normal rat kidney displayed low-level expression of ORP150 antigen in the renal medulla (Fig. 1J
), whereas no signal was detected in the cortex (Fig. 1K
). After I/R, ORP150 antigen was markedly induced in renal tubules within the medulla (Fig. 1L
), as well as in portions of renal tubules in the cortex (Fig. 1M)
.
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2. ORP150 suppresses cell death in renal epithelial cells
Exposure of MDCK cells, a renal tubular epithelial cell line, to hypoxia caused expression of ORP150 antigen; the latter increased by 12 h and reached a maximum between 24–48 h. Incubation of MDCK cultures in presence of high salt (NaCl, 300 mM) also induced ORP150 antigen. Combination of hypoxia and hyperosmolarity appeared to potentiate ORP150 expression to levels greater than that observed with either stimulus alone. Stable transfectants of MDCK cells were made with antisense or sense constructs of human ORP150. After exposure of these stably transfected cell lines to hypoxia (24 h), antisense transfectants demonstrated detectable, but low levels of ORP150 antigen; vector-alone transfectants showed higher levels of ORP150; and sense transfectants displayed highest levels of ORP150. To evaluate vulnerability of MDCK stable transfectants to hypoxia and hyperosmolar stress, cultures were exposed to hypoxia in presence of NaCl (500 mM) for 36 h, and cell death was evaluated by release of LDH and induction of apoptosis by activation of caspase-3. Results demonstrate increased cell death and activated caspase-3 in antisense transfectants and lowest levels of cell death/activated caspase-3 in sense transfectants. These data indicate a correlation between expression of ORP150 and cellular resistance to hypoxia/hyperosmolar-induced cell death.
3. Furosemide suppressed expression of ORP150 transcripts by unilateral nephrectomy and I/R
The effect of furosemide (a loop diuretic) on ORP150 expression after unilateral nephrectomy and I/R was assessed. Unilateral nephrectomy induced ORP transcripts in the remaining kidney, with peak expression 8–12 h after the procedure. Unilateral I/R also caused prominent up-regulation of ORP150 mRNA, in this case in both kidneys with the most striking effect on the ipsilateral side. Pretreatment with furosemide suppressed expression of ORP150 mRNA in both unilateral nephrectomy and I/R. This suggests the possibility that increased ORP150 mRNA observed in both contralateral/ipsilateral kidneys after I/R may be due to osmotic stress, at least in part.
4. ORP150 suppressed renal dysfunction in a murine model after I/R, by protecting cell viability in TAL
Mice with genetically manipulated expression of ORP150 were used to assess the effect of ORP150 on renal function following I/R injury. For these studies, mice were prepared by right nephrectomy followed seven days later by occlusion of the left renal pedicle for 45 min (I or ischemia) and reperfusion (R). Tg ORP150 mice displayed relative resistance to renal dysfunction, based on the blunted rise in serum creatinine and serum/blood urea nitrogen compared with ORP150+/– animals. Caspase-3 activity was assessed in renal tissue as an index of programmed cell death. Highest levels were observed in ORP150+/– animals, intermediate levels in ORP150+/+ mice, and lowest levels in Tg ORP150 mice.
To further localize the protective effect of ORP150 expression on the kidney, immunohistochemical studies were performed with an antibody selective for activated caspase-3 and renal tubular markers. In the renal cortex, there was no significant difference in the proportion of nuclei staining positively with caspase-3 antibody comparing mice of each genotype (Fig. 2
A). In medulla, caspase-3-positive nuclei were most frequently observed in ORP150+/– mice, whereas they were least abundant in Tg ORP150 animals (Fig. 2A
). Since caspase-3-positive signals identified by this method were likely to include deteriorating cells in casts, we further sought to map distribution of potentially apoptotic cells in portions of the renal segment (Fig. 2B-N
). Although there was no significant difference in the percentage of caspase-3-positive nuclei in proximal and distal renal tubules between the three genotypes, there were differences in TAL based on colocalization with THP (Fig. 2B
). These data suggest that overexpression of ORP150 in the kidney enhances cellular viability in response to ischemic challenge, especially in TAL.
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CONCLUSIONS AND SIGNIFICANCE
We have identified ORP150 as a stress protein expressed in I/R-mediated injury to the kidney. ORP150 appears to have a cytoprotective effect on renal epithelial cells both in vitro and in vivo in response to I/R and hyperosmolar stress. Though the precise mechanism through which ORP150 exerts its cytoprotective effect in TAL remains to be defined, it is likely to involve its chaperone-like properties in the ER. Since cytoprotective effects of ORP150 (an ER chaperon) were focused on TAL, we suggest that maintenance of ER function is an essential component of a successful stress response in this portion of the nephron in acute renal failure.
Data presented in this manuscript indicate that ORP150 is also induced in response to hyperosmolar stress, and this is accentuated by superimposed oxygen deprivation. Our data suggest that both ischemic and osmolar stress targets a cellular organelle (ER) resulting in an accumulation of immature and unfolded proteins inside (Fig. 3
). Resistance of MDCK cells to this complex environmental challenge is dependent at least in part on ORP150.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1161fje; doi: 10.1096/fj.03-1161fje
1 These authors contributed equally to this work. 
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-activated caspase-3 antibody, followed by staining with DAPI. Nuclei staining positive for activated caspase-3 were counted in the cortex and medulla, and values were expressed as % positive nuclei (total nuclei were determined by DAPI staining). Mean ± SD is shown (n=6); **P <0.05 vs. observations in ORP150+/+ mice, by multiple comparison analysis. B) Sections were double-stained with segment markers and 