Protective Effects And Mechanism Of Intermedin Up-regulation In Rat Renal Ischemia/Reperfusion Models

BackgroundIntermedin (IMD) is a newly discovered peptide that belongs to calcitonin gene-related peptide(CGRP). It has been proved that IMD has protective effects on ischemia/reperfusion (I/R) in isolated rat hearts, whereas patho-physiology function of IMD in renal I/R is not clear.ObjectiveIn the present study, we aim to design and construct eukaryotic expression vector encoding rat IMD gene and to transfect it into kidney tissue with ultrasound-mircobubbles in order to observe effects of IMD on renal I/R in rat models and to investigate the mechanisms of IMD protective function in renal I/R.Methods1. Construction and identification of eukaryotic expression plasmid encoding rat IMD geneFirstly, full-length rat IMD gene in gene bank was retrieved. The cloning plasmid named as pGH-IMD-X1491G was synthetized and then sequencing analysis was performed. Secondly, pGH-IMD-X1491G was linearrizated by HindⅢand EcoRI enzyme digestion and then was ligated to pCDNA3.1(+) eukaryotic expression vector. Finally, the recombinant plasmid designated as IMD-pCDNA 3.1 was confirmed by restrictive enzyme digestion.2. Effects of IMD in renal I/R rat modelsA total of 24 rats were divided into four groups each consisting of six animals. GroupⅠunderwent right nephrectomy one week prior to the exposure of left renal pedicles, but did not receive any ischemia/reperfusion. GroupⅡunderwent right nephrectomy one week prior to left renal I/R surgeries. GroupⅢunderwent right nephrectomy and left renal IMD-pCDNA3.1(+) transfection by ultrasound-mircobubbles and renal I/R surgeries were performed one week after gene transfection. Group IV animals were treated the same way as group III except that empty control vector was transfected. All the animals were killed at the end of 24 h of reperfusion before the sera and kidney tissues were kept for detection. Serum levels of ALT, AST, BUN and creatinine were detected by Beckman automatic biochemistry analyzer. The mRNA expressions of IMD in the kidneys were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The protein expressions of IMD in the kidneys were detected by Western blotting. Kidney formaldehyde-fixed paraffin sections were stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) by standard methods and then histological changes were analyzed semiquantitatively.3. Researches into the mechanism of IMD protective function of rat renal I/RThe mRNA expressions of endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in the kidneys of the four groups were determined by RT-PCR. The protein expressions of eNOS, iNOS, nNOS, ET-1 and TNF-a in the kidneys were semiquantitatively analyzed by Western blotting.Results1. Construction and identification of eukaryotic expression plasmid encoding rat IMD geneRestriction enzyme digestion and sequencing analysis showed that eukaryotic expression plasmid of IMD-pCDNA3.1(+) had been constructed successfully.2. Effects of IMD in renal I/R rat modelsThe serum levels of BUN and Cr in rats of group III were obviously lower than those of groupⅡand groupⅣ(P<0.05), while the differences of ALT and AST levels among the four groups were not evident. Compared with group II and group IV, the mRNA expressions of IMD in kidneys of group III were up-regulated significantly by 60.7%,66.1% and the protein expressions of IMD in kidneys increased significantly by 51.4%,55.9%. I/R animals in groupⅡ&IV exhibited serious tubular cellular vacuolization and necrosis, loss of brush border, cast formation and tubular dilatation, which were significantly lessened in the ultrasound-mediated IMD gene transfer group. When compared with IMD-treated I/R group, rats in groupⅡandⅣproduced a significant increase in total severity score (P<0.05)3. Researches into the mechanism of IMD protective function of rat renal I/RCompared with those in groupⅡ, we noticed higher mRNA and protein expressions of eNOS in groupⅢ, while the mRNA and protein expressions of iNOS in groupⅢwere obviously lower compared with those in group of I/R without IMD gene treatment (P<0.05). Meanwhile, there were no significant differences in the mRNA and protein expressions of nNOS among groupⅡ,ⅢandⅣ. Moreover, the protein expressions of ET-1 and TNF-αin groupⅢwere obviously lower compared with those in group of I/R without IMD gene treatment (P<0.05)Conclusions1. Eukaryotic expression plasmid of IMD-pCDNA3.1(+) was constructed successfully.2. It is safe and effective to transfect IMD-pCDNA3.1(+) eukaryotic expression plasmid into kidney tissue with ultrasound-mircobubbles as it could lead to the up-regulation of IMD in the local site of kidney tissue. 3. Up-regulation of IMD prior to renal I/R could protect kidney against tubulointerstitial damage and renal dysfunction in I/R rat models.4. IMD gene in kidneys of rats can promote the expressions of eNOS and attenuate over-expressions of iNOS, ET-1 and TNF-αfollowing I/R, thus protect kidneys against severe damages in I/R rat models.