HGF gene transfer increases kidney graft survival

Abstract

The introduction of cyclosporine (CsA) into clinical practice has resulted in marked improvement in the short-term outcome of organ transplantation, and 1-year survival of renal allografts has improved significantly. However, the annual rate of kidney graft loss caused by chronic allograft nephropathy (CAN) has remained stable over last decade. CAN may result from perioperative ischemia at the time of transplantation. Furthermore, chronic CsA nephrotoxicity may progress to an irreversible renal lesion characterized by tubular atrophy, striped interstitial fibrosis, hyalinosis of the afferent arteriole, and progressive renal impairment. Recent report demonstrated the therapeutic effects of hepatocyte growth factor (HGF) in preventing CAN using a well-established rat CAN model. They showed that HGF treatment during the initial 4 weeks after engraftment prevented onset of CAN and associated death and provided longlasting benefit in terms of graft survival. Exogenous HGF is very unstable in the blood circulation due to the rapid clearance by the liver. To circumvent this problem, we developed a new gene transfer system by electroporation in vivo. Therefore, this gene transfer approach represents a useful technique to investigate the therapeutic potential of HGF gene transfer for long-term survival of kidney allograft. The goal of the present study was to assess the renoprotective potential and safety of HGF gene transfer using a porcine kidney transplant warm ischemia injury model or CsA nephrotoxicity model. In the first set of experiments, following left porcine kidney removal, 10 minutes of warm ischemic injury was intentionally induced. Next, the HGF expression vector or vehicle was infused into the renal artery with the renal vein clamped ex vivo, and electric pulses were discharged using bathtub-type electrodes. Kidney grafts were then transplanted after removing the right kidney. Histopathologic examination of vehicle-transfected kidney transplant revealed initial tubular injury followed by tubulointerstitial fibrosis. In contrast, HGF-transfected kidneys showed no initial tubular damage and no interstitial fibrosis at 6 months posttransplant. In the next set of experiments, CsA was subcutaneously administered daily under low sodium diet, and HGF gene was transferred into skeletal muscle by electroporation on days 7 and 14. We also examined the antiapoptotic mechanism of HGF using human proximal tubular epithelial cells. HGF gene transfer rescued CsA-induced initial tubular injury, and suppressed interstitial infiltration of endothelium-1 (ED-1)-positive macrophages in CsA nephrotoxicity. In addition, HGF significantly inhibited tubular cell apoptosis, and increased the number of proliferating tubular epithelial cells. In vitro studies suggest that HGF executes the antiapoptotic function by enhancing the phosphorylation of Akt and Bcl-2. Northern blot analysis demonstrated that HGF gene transfer suppressed cortical mRNA levels of transforming growth factor-β (TGF-β). Consequently, HGF gene transfer significantly reduced a striped interstitial phenotypic alteration and fibrosis. We conclude that electroporation-mediated HGF gene transfection protects the kidney against graft injury.