Intermedin protects against renal ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress

Abstract

Background: Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. Endoplasmic reticulum stress (ERS) has been implicated in the pathology of renal ischemia/reperfusion (IRI). In the present study, we investigated whether IMD could reduce ERS damage after renal ischemia.

Methods: The kidneys of SD rats were subjected to 45 min of warm ischemia followed by 24 h of reperfusion. The hypoxia/reoxygenation(H/R) model in NRK-52E cells consisted of hypoxia for 1 h and reoxygenation for 2 h. IMD was over-expressed in vivo and in vitro using the vector pcDNA3.1-IMD. The serum creatinine concentration and lactate dehydrogenase (LDH) activity in the plasma were determined. Histologic examinations of renal tissues were performed with PAS staining. Real-time PCR and Western blotting were used to determine the mRNA and protein levels, respectively. Additionally, ER staining was used to detect the ERS response.

Results: In the rat renal IRI model, we found that IMD gene transfer markedly improved renal function and pathology and decreased LDH activity and cell apoptosis compared with the kidneys that were transfected with the control plasmid. IMD significantly attenuated the ERS stress parameters compared with IRI group. Indeed, IMD down-regulated glucose-regulated protein 78 (GRP78), C/EBP homologous protein(CHOP), and caspase 12 protein and mRNA levels. Moreover, in the NRK-52E cell H/R model, IMD overexpression prevented the apoptosis induced by H/R. Furthermore, IMD ameliorated the ER structural changes and concomitantly decreased the levels of GRP78, CHOP and caspase-12.

Conclusion: This study revealed that IMD protects against renal IRI by suppressing ERS and ERS-related apoptosis.

Fig. 1

The transfection efficiency of IMD by ultrasound-mediated gene delivery into the Kidney in vivo and by FuGENE HD into NRK-52E cells in vitro. a Representative IMD protein expression measured by Western blot in rats, (b) representative IMD protein expression measured by Western blot in NRK-52E cells, (c) Quantitative analysis of IMD by Western blots in rats, (d) Quantitative analysis of IMD by Western blots in NRK-52E cells, (e) representative IMD mRNA expression measured by RT-PCR in rats, (f) representative IMD mRNA expression measured by RT-PCR in NRK-52E cells, (g) densitometric quantifications of band intensities from RT-PCR for IMD/β-actin in rats, (h) densitometric quantifications of band intensities from RT-PCR for IMD/β-actin in NRK-52E cells. M: DL500 DNA marker, Data in bar graphs are means ± SD, n = 6.* P < 0.05 vs. sham group or control group; # P < 0.05 vs. IRI group or H/R group, respectively

Fig. 2

IMD ameliorated renal injury in rats after IRI treatment. a Serum creatinine concentration, (b) the LDH activity in different IRI treatment groups. c - f Renal pathomorphological changes after renal ischemia-reperfusion injury (IRI), Original magnification, ×400; (c) Sham groups, (e) IRI groups, (e) IRI + empty plasmid groups, (f) IRI + IMD groups, Magnification:×400 (g) semiquantitation of the morphological changes by histological grading system. Data in bar graphs are means ± SD, n = 8. * P < 0.05, **P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 vs. IRI group, respectively

Fig. 3

IMD suppressed cell apoptosis triggered by renal IRI. a - d Determination of tubular cell apoptosis in situ by terminal deoxynu-cleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) assay. (×200) (a) Sham groups, (b) IRI groups, (c) IRI + empty plasmid groups, (d) IRI + IMD groups, Magnification:×400 (e) Determination of positive tubular cells in the TUNEL assay. Data in bar graphs are means ± SD, n = 8. **P < 0.01 vs. sham group; ## P < 0.01 vs. IRI group

Fig. 4

Effects of IMD on markers of endoplasmic reticulum stress (ERS) in IRI rats. a Representative protein expression of GRP78, CHOP, caspase-12, and β-actin as a control; Quantitative analysis of (b) GRP78, (c) CHOP, and (d) caspase-12 by Western blots. e The mRNA expression levels of GRP78, CHOP and caspase-12 were determined by real time-PCR. Data in bar graphs are means ± SD, n = 8. * P < 0.05, **P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 vs. IRI group, respectively

Fig. 5

IMD inhibited ERS-related apoptosis in NRK-52E cell of H/R injury. a - e Cell apoptosis measured by flow cytometry. a control, b Tm, c H/R, d H/R + pcDNA3.1, e H/R + IMD, f The apoptosis ratio in different H/R treatment groups. g ER morphology (blue) in NRK-52E cells loaded with Dapoxyl according to the kit. Data in bar graphs are means ± SD, n = 6. **P < 0.01 vs. control group; ## P < 0.01 vs. Tm or H/R group, respectively

Fig. 6

IMD inhibited tunicamycin (Tm) and H/R-induced (ERS) in vitro. a Representative protein expression of GRP78, CHOP, caspase-12, and β-actin as a control; Quantitative analysis of (b) GRP78, (c) CHOP, and (d) caspase-12 by Western blots. e The mRNA expression levels of GRP78, caspase-12, CHOP were determined by real time-PCR. Data in bar graphs are means ± SD, n = 6. * P < 0.05, **P < 0.01 vs. control group; # P < 0.05 vs. Tm or H/R group, respectively