Experimental Study Of The Protection From Intermedin In Rat Renal Ischemia Reperfusion Injury

Being a newly-found endogenous cardio-renal-protective substance, Intermedin (IMD) belongs to the calcitonin gene-related peptide (CGRP) superfamily; its renal-protection mechanism is still unknown. Applying the methods of both cytobiology and molecular biology, this study is designed to explore the physiological and pathological significance of IMD in renal ischemia-reperfusion injury(IRI). This study includes two parts:1. The changes of IMD and its receptors in rat renal ischemia reperfusion injuryObjective The rat models of renal ischemia reperfusion injury(IRI) were established to observe the changes of IMD and its receptors after IRI, and explore the functions of IMD in renal IRI. Methods Eighteen male Wistar rats were randomly divided into sham-operated,ischemia and IRI groups. IRI rat models were made by clamping both renal arteries for 45min and reperfusing for 12 hours. IRI models were evaluated through measuring blood urea nitrogen(BUN) and serum creatinine(SCr) concentration, and through semi-quantitated anaylsis of renal histological changes. The contents of IMD and ADM in plasma and renal tissue were assessed by radioimmunoassay (RIA). The mRNA expressions of IMD, ADM, CRLR and RAMPs in the kidneys were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR). The changes of protein expressions of IMD and CRLR in the kidneys were detected by Western blotting.Results⑴Compared with the sham ones, BUN and SCr in IRI rats increased significantly(P<0.05). Kidneys pathologic changes of IRI rat models were significant, as shown in tubule vacuolar degeneration or necrosis, loss of brush border; while there was little change in glomerulus. Semi-quantitated analysis suggests there was significant difference between IRI groups and the other two groups. (the sham-operated groups, P<0.05 and the ischemia groups, P<0.01). The results go with the changes of acute renal injury, so models are successfully established.⑵Compared with sham-operated and ischemia groups, the IMD contents of serum and renal in IRI group increased by 53.76%, 40.01% and 58.34%, 42.97% (P<0.05), and serum ADM showed a marked increase by 143.22%, 101.96% (P<0.05).⑶Compared with sham-operated group, the mRNA expressions of IMD, ADM, CRLR, RAMP1, RAMP2, RAMP3 in kidneys of IRI group were all up-regulated significantly by 22.1%(P<0.05, 41.1%(P<0.01), 32.8%(P<0.01), 33.7%(P<0.01), 35.9%(P<0.05), 29.4%(P<0.01), and the protein expressions of IMD and CRLR in kidneys increased significantly by 80.9%, 68.4%(P<0.001).Conclusion In IRI group, levels of IMD in blood plasma and kidney increased significantly; and consistently there appeared marked increases of the mRNA expressions of IMD and of its receptors, and the protein expressions of IMD and CRLR in kidneys. These results suggest that IMD may have participated in the process of renal IRI, and may have important physiological and pathological significance in terms of kidney protection.2. The influence of IMD exerted on the hypoxia/reoxygenation model of rat renal tubular epithelial cells and its mechanismObjective The hypoxia/reoxygenation(H/R) injury models of rat renal tubular epithelial cells (NRK-52E), which were transfected the eukaryotic expression vector of rat IMD, were established to simulate IRI in vivo, to investigate the molecule mechanism of IMD cytoprotection.Methods⑴To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 1h and then with reoxygenation for 1.5h. The models were evaluated by detecting living cell count, cell viability and the activity of lactate dehydrogenase (LDH) in the culture medium.⑵The full length IMD cDNA, which was synthesized artificially, was connected with pMD19-T Simple vector to form a cloned vector. This cloned vector was sub-cloned into pIRES2-EGFP to construct an eukaryotic expression vector, which is named pIRES2-EGFP/IMD. Restriction enzyme digestion and sequencing analysis were adopted to test the recombinant plasmids.⑶NRK-52E cells were transfected by transfection complex comprising optimal proportion of pIRES2-EGFP/IMD plasmid and Fugene HD reagents. Transfection efficiency was tested by observation for EGFP made by fluorescent microscope and flow cytometery (FCM) after 48h. Total RNA and protein was extracted from EGFP positive NRK-52E cells. IMD mRNA and protein expression level was evaluated by RT-PCR and Western-blotting. The cell groups with high transfection efficiency were screened for two weeks or so by incubating with medium containing G418(300μg/ml), and the positive cloning NRK-52E cells of stable expression IMD obtained. The expression of IMD protein in IMD plasmid group after screening was evaluated by Western-blotting.⑷The NRK-52E cells were divided into 7 groups. One of them was control group; the other six model groups were exposed to H/R condition, following the intervention of single H/R, primitive vector, highly expressed IMD vector, NADPH oxidase inhibitor(DPI), cyclic adenosine monophosphate(cAMP) analogue (8-Br-cAMP), PKA inhibitor(H-89), respectively. The contents of LDH and cAMP in the culture medium and the levels of NADPH oxidase, superoxide dismutase(SOD), malondialdehyde(MDA) and reactive oxygen species(ROS) in cells were detected to observe the influence of IMD on H/R injury and its molecule mechanism.Results⑴After hypoxia 1h and reoxygenation 1.5h, cell count and cell viability decreased significantly and the activity of LDH increased significantly (P<0.05); the model was established successfully.⑵Restriction enzyme digestion and sequencing analysis showed that pMD19-T Simple/IMD cloning vector and pIRES2-EGFP/IMD eukaryotic expression vector had been constructed successfully.⑶NRK-52E cells was transfected with the optimal transfection proportion of pIRES2-EGFP/IMD to Fugene HD reagent as 3μg: 12μl, transfection efficiency evaluated by fluorescence microscope and FCM showed that the transfection efficiency increased with the prolongation of time and reached its peak 71.34±6.24% at 48h(P<0.01). The result of RT-PCR and Western blotting showed the expression levels of IMD mRNA and protein in IMD plasmid group were increased significantly (P<0.01 and P<0.001, respectively). The positive transfected cells after G418 screening were found clone-like proliferation on the third day, and the untransfected cells died in large bulk on the fifth day. Then the positive transfected cells increased gradually; and the positive cloning cells blending into slices were found on the fourteenth day, which occupied 60-70% of the total cell number. The result of Western blotting showed the expression of IMD protein in IMD plasmid group after screening increased significantly, which indicate the IMD expression of the cell is stable.⑷In NRK-52E cells following the seven treatments, the changes of detected index were shown:①Contrasted with the control group, the level of LDH of all model groups increased significantly (P<0.01); but the extent of increase in IMD plasmid group and 8-Br-cAMP group were smaller than that in H/R group(P﹤0.05).②Contrasted with the control group, the level of cAMP of all model groups also increased significantly(P<0.01), and IMD plasmid + H-89 group and 8-Br-cAMP group were exceptionally noticeable; when compared with H/R group, the two groups were the most remarkably increased groups(P﹤0.01), IMD plasmid group increased mildly and DPI group decreased(P﹤0.05).③Contrasted with the control group, the activity of NADPH oxidase of all model groups increased significantly except DPI group(P<0.01), but the extent of increase in 8-Br-cAMP group was the smallest. Compared with H/R group, the levels of NADPH oxidase of 8-Br-cAMP group and DPI group decreased by 33.92%(P﹤0.05) and 52.54%(P﹤0.01), respectively.④The content of MDA increased in H/R group (P﹤0.01), primitive plasmid group (P﹤0.01), IMD plasmid + H-89 group (P﹤0.05) and DPI group (P﹤0.01), but decreased in IMD plasmid group, IMD plasmid + H-89 group, 8-Br-cAMP group and DPI group(P﹤0.05) compared with H/R group.⑤The content of ROS increased in H/R group, primitive plasmid group, IMD plasmid + H-89 group (P﹤0.05), but decreased in IMD plasmid group compared with H/R group(P﹤0.05).⑥The content of SOD increased in IMD plasmid group, IMD plasmid + H-89 group and DPI group(P﹤0.05), but it changed little in the other four groups.Conclusion In H/R model of NRK-52E, IMD could increase the level of cAMP via cAMP/PKA access, inhibit the activity of NADPH oxidase, decrease the production of ROS, upgrade SOD, alleviate the cell oxidative damage, regulate the oxidative and anti-oxidative balance. All these suggest that IMD, as an endogenous antioxidant, plays a protective role in cell protection during H/R-induced oxidative injury.