A novel therapy to attenuate acute kidney injury and ischemic allograft damage after allogenic kidney transplantation in mice

Abstract

Ischemia followed by reperfusion contributes to the initial damage to allografts after kidney transplantation (ktx). In this study we tested the hypothesis that a tetrapeptide EA-230 (AQGV), might improve survival and attenuate loss of kidney function in a mouse model of renal ischemia/reperfusion injury (IRI) and ischemia-induced delayed graft function after allogenic kidney transplantation. IRI was induced in male C57Bl/6N mice by transient bilateral renal pedicle clamping for 35 min. Treatment with EA-230 (20-50mg/kg twice daily i.p. for four consecutive days) was initiated 24 hours after IRI when acute kidney injury (AKI) was already established. The treatment resulted in markedly improved survival in a dose dependent manner. Acute tubular injury two days after IRI was diminished and tubular epithelial cell proliferation was significantly enhanced by EA-230 treatment. Furthermore, CTGF up-regulation, a marker of post-ischemic fibrosis, at four weeks after IRI was significantly less in EA-230 treated renal tissue. To learn more about these effects, we measured renal blood flow (RBF) and glomerular filtration rate (GFR) at 28 hours after IRI. EA-230 improved both GFR and RBF significantly. Next, EA-230 treatment was tested in a model of ischemia-induced delayed graft function after allogenic kidney transplantation. The recipients were treated with EA-230 (50 mg/kg) twice daily i.p. which improved renal function and allograft survival by attenuating ischemic allograft damage. In conclusion, EA-230 is a novel and promising therapeutic agent for treating acute kidney injury and preventing IRI-induced post-transplant ischemic allograft injury. Its beneficial effect is associated with improved renal perfusion after IRI and enhanced regeneration of tubular epithelial cells.

Figure 1. IRI was performed and 24h after surgery EA-230 or vehicle treatment was given twice daily i.p. for four consecutive days.

The majority of vehicle treated mice died within four days after IRI (A). EA-230 treated mice receiving doses between 30 and 50mg/kg survived significantly better than vehicle treated mice (***p<0.005). Renal function measured by s-creatinine (B) showed the initial increase and normalized over time in the surviving mice of all groups. Renal blood flow (C, PAH clearance), glomerular filtration rate (D, inulin clearance) and urine output (E) were significantly higher in EA-230 treated mice (*p<0.05).

Figure 2. Acute tubular necrosis (A-C) two days after IRI affected ∼25% of tubuli in the vehicle treated mice and was less in the EA-230 treated mice (A-C, magnification 200 fold).

The number of Ki-67 positive tubular epithelial cells as marker of proliferation (red staining) was significantly higher in EA-230 treated kidneys two days after IRI (D-F, magnification 200fold, the autofluorescent kidney tissue appears green).

Figure 3. Isogenic transplantation (blue) revealed 90% survival for four weeks and normal renal function (B).

After allogenic kidney transplantation 90% of the vehicle treated mice died within seven days after nephrectomy of the contralateral native kidney of the recipients at day 4. In contrast 70% of EA-230 treated mice were still alive one week after ktx. 30% of the recipients treated with EA-230 achieved four week survival whereas all vehicle treated recipients died within the first two weeks. Renal function (B) deteriorated at day six in both allogenic ktx groups but was significantly better in EA-230 (green columns, ***p<0.005) than in vehicle treated allograft recipients (red columns).

Figure 4. PAS stained isografts (A) and allografts with vehicle treatment (B) or EA-230 50mg/kg (C) treatment six days after transplantation.

Isografts had no signs of acute rejection or acute tubular necrosis. Vehicle treated allografts showed severe ATN with 50% of the tubuli affected and Banff 1A acute rejection with interstial infiltrates, tubulitis and gomerulitis (B). EA-230 treated allografts (C) showed mild ATN with ∼10% of tubuli affected and borderline rejection (magnification 200 fold, D semiquantitative score for ATN).